Abstract

The eighth annual RNAi international conference and exhibition,
RNAi2013 was hosted at St Hilda’s College, Oxford,
UK (19-21 March 2013), and provided a platform for congregation
of researchers with both academic and industrial
backgrounds to share and discuss their most recent work in
the fast advancing field of RNAi. RNAi has been recognised
as a fundamental method for functional genomic investigations
and has great potential as a therapeutic intervention
for several human diseases. RNA-induced gene expression
inhibition mechanisms were discussed for both the benefit
of research and clinical therapeutics. The conference
conveyed an impressive series of presentations given by
national and international RNAi research leaders. Additionally,
research posters were exhibited for the entirety of the
conference. Furthermore, technology workshops were provided
by Sigma-Aldrich, Eupheria Biotech GmbH and Carl
Zeiss enabling conference attendees to learn about their
most advanced RNAi expertise. These companies also participated
in a trade exhibition along with Exiqon to promote
the latest commercial RNAi products.

Rnai Developments

Professor Kaz Taira (University of Tokyo, Japan) was invited
as a special guest speaker and opened the conference with
a presentation describing the recent discovery that siRNA
strand antagonism is the major cause of reduced siRNA
potency when compared with the potency of shRNA. RNAi
activity of siRNA is reduced compared to shRNA due to the
sense RNA strand negatively regulating RNAi. By modifying
the relative sense and antisense components of duplex
siRNA during expression, improved potency of siRNA in
target gene RNAi was achieved (Jin et al, 2012). Furthermore,
Taira identified DEAD-box helicase 3 (DDX3) using
a short hairpin RNA-expression library, as a fundamental
component of the RNAi pathway. DDX3 was found to colocalise
with Argonaut2 (Kasim et al, 2013).

Dr Laure-Alix Clerbaux (Université catholique de Louvain)
elucidated the mechanism used by cells to maintain cholesterol metabolism. Clerbaux explained that the primary transcript
of sterol regulatory element binding protein (SREBP) 2 contains
not only the genetic code for a sterol sensing transcription
factor which promotes transcription of numerous genes
involved in cholesterol and fatty acid synthesis, but also holds
the highly conserved intronic miR-33. miR-33 was shown to
target and successfully down-regulate activity of the cholesterol
export pump, ABCA1, which considerably reduced
cellular cholesterol export and hence increased cellular cholesterol
concentration. The SREBP is triggered during low
cellular cholesterol levels; it was revealed therefore, that
miR-33 interacts with the gene in which it is located to maintain
normal cellular cholesterol levels (Gerin et al, 2010).

Nanoparticle Delivery of Rnai Therapeutics

Due to their sensitivity to enzymatic degradation, large
negative charge, high molecular weight, and rapid plasma/
renal clearance, ncRNA therapeutics are notoriously difficult
to deliver into mammalian cells. Hence, successful
delivery of ncRNA is a great challenge within the RNAi
therapeutic field. In an attempt to overcome these issues,
numerous non-viral nanoparticles administered by systemic
intravenous injection have been developed recently to
enable therapeutic use of synthetic ncRNA for a number of
RNAi applications.

Professor Gilles Divita (Centre National de la Recherche
Scientifique, France) described NANOVEPEP technology
which involves nanoparticle self-assembly around the
siRNA, aided by electrostatic and hydrophobic interactions
between short amphipathic CADY peptides. NANOVEPEP
technology is particularly advantageous since delivery of
siRNAs into specific cell targets is possible without initiating
an inflammatory response (Konate et al, 2012).

Anti-Cancer Rnai Therapeutics

Since cancer is caused by accumulation of genetic abnormalities,
nucleic acid medicines are an obvious therapeutic
choice and are predicted to have the most potential for
success. Many nucleic acid therapies are currently being
developed; a selection of the latest RNAi associated cancer
therapies were presented at RNAi2013.

Chemoresistance is a major limitation of drugs currently
used to treat cancer and results in significantly reduced survival
rates. Dr David Carter (Oxford Brookes University,
UK) described characterisation of miRNA levels in ovarian
cancer cell lines that are resistant or sensitive to cisplatin
treatment. Through loss or gain of function experimentation
a miRNA and coding gene pair were identified that can contribute
to cisplatin resistance during carcinogenesis.

Professor Achim Aigner (University of Leipzig, Germany)
demonstrated that Pim-1 activity, previously linked with
poor prognosis, is fundamental to signal transduction in
colon carcinoma and glioblastoma cells and is regulated by
miR-15b and miR-33a. Knockdown resulted in anti-tumour
effects; treated cells also became more sensitised to 5-FU
(Thomas et al, 2012).

Dr Nigar Babae (University Medical Centre Utrecht, the
Netherlands) presented results that identified a novel antiangiogenic
miRNA using a lentiviral miRNA expression
library. Following local delivery by electroporation in a
Neuro2A mouse tumour model, tumour growth rate diminished
by 50% and tumour vascularisation was prevented.
The identified miRNA was discovered to regulate expression
of approximately 2500 genes; two of these genes were
further investigated due to their role in vascularisation.

The most difficult challenge for anti-viral drug design is the
prevention of drug-resistant strains. In order to prevent such
occurrences, many strategies are currently being developed;
with the most sophisticated methods involving RNAi.

Dr Susanna Obad (Santaris Pharma, Denmark) described the
development of miravirsen, a drug to treat chronic hepatitis
C virus (HCV) infection. Miravirsen is a locked nucleic acid
(LNA) and DNA mixmer oligonucleotide that targets miR-
122. miR-122 acts as a liver specific host factor during HCV
infection. After success in chronically HCV infected chimpanzees
(Elmén et al, 2008), the first clinical trial involving
miRNA inhibition was launched. Phase IIa clinical trials
have recently been completed which concluded that when
used to treat patients suffering with chronic HCV genotype
1 infection, miravirsen exhibited extended dose-dependent
decrease in HCV RNA levels with no evidence of resistant
viral strains (Janssen et al, in press). These results are promising
for the development of LNA-antimiR oligonucleotides
for targeting of additional miRNAs that contribute to pathogenesis
in humans.

Rnai Treatment for Disease

DMD-associated miRNAs (dystromiRs) are potential biomarkers
for Duchenne Muscular Dystrophy (DMD). Dr
Tom Roberts (University of Oxford, UK and the Scripps
Research Institute, USA) showed that differing levels of
dystromiRs were detected between different types of skeletal
muscle (Roberts et al, 2012). Additional results suggest
that dystromiRs are transported in the circulation bound to
protein/lipoprotein complexes to protect them from nuclease
activity.

Dr Kathia Zaleta-Rivera (Stanford University, USA) discussed
allele specific oligonucleotide treatment for hypertrophic
cardiomyopathy (HCM) anticipated to salvage
expression of the wild-type allele enabling recovery of cardiomyocyte
functionality. siRNA and short hairpin (shRNA)
treatments were identified for 2 mutations associated with
HCM. Models for each mutation have been developed to
measure changes in cell contractility and force generation
after treatment with shRNAs.

Professor Paul Holvoet (University of Leuven, Belgium)
described the relationship between obesity and atherosclerosis
identified by shared expression of a collection of
miRNAs. The identified miRNAs were found to regulate
adipocyte differentiation, oxidative stress, inflammation,
and angiogenesis in adipose tissues of obese patients and
vascular tissues of atherosclerosis patients. Repression of
specific miRNAs was discovered to induce oxidative stress
and inflammation. To complete the vicious circle, reduced
levels of these miRNAs were recognised to contribute to
development of obesity a condition that in itself increases
the risk of development of atherosclerosis. The discovery
of miRNA containing monocyte-derived micro-particles
that participate in intercellular communication within and
between adipose and atherosclerotic vascular tissues, were
also discussed (Hulsmans et al, 2011).

Oligonucleotides in Rnai

The conference keynote speaker Dr Mike Gait (MRC Laboratory
of Molecular Biology, Cambridge, UK) presented
recent work involving development of peptide nucleic acids
(PNA) anti-miRs which rapidly inhibit miR-122 in liver cells
without the participation of transfection agents (Torres et al,
2012). Following uptake anti-miRs target miRNAs within or
in relation to the endosomal compartment and strong dose
dependent miR-122 inhibition has been observed for phosphorothioated
oligonucleotide counterparts (Torres et al,
2011). These results are promising for the development of
both diagnostic markers and anti-miR therapeutics for a
wide range of genetic disorders.

Dr Dmitry Samarsky (RiboBio, China) reported the design
of a sophisticated type of RNAi molecule composed of single
chain oligonucleotide which has both 5′ and 3′ targeting
regions which mediate self-dimerisation with partial complementarity.
The loop containing-RNA duplex molecule
has been shown to enter and activate RISC and is promising
for the future design of single-stranded oligonucleotide
therapies (Lapierre et al, 2011).

Minimalisation of Off-Target Effects

One of the greatest limitations of siRNA-based gene silencing
is the incidence of sequence-specific off-target effects.
These adverse interactions are often not foreseen because
siRNAs can induce gene silencing through association
with regions of the genome with only partial complementarity.
During these incidences the siRNA functions as a
miRNA inhibiting gene expression by destabilising mRNA
or blocking transcription. Therefore off-target interactions
are a major consideration during siRNA design. Dr Michael Hannus
(Intana Bioscience GmbH, Germany) described a
possible resolution which employs siPools which contain up
to 60 specifically selected siRNA molecules. Within a siPool
each siRNA is retained at a low concentration so off-target
interactions are reduced to such a degree that they lie below
the lower limit of detection.

Technical Focus

In order for the ever-evolving field of RNAi to grow, the
most advanced and innovative technology needs to be developed
and utilised by researchers worldwide. Presentations
and exhibitions by Sigma-Aldrich, Eupheria Biotech GmbH
and Carl Zeiss described their latest products and/or techniques
that may assist scientists in cutting-edge research.

Dr Steven Thompson (Sigma-Aldrich, UK) described
CompoZr® Zinc Finger Nucleases (ZFNs) which facilitate
genome manipulation through site-specific mutagenesis
by generating double-strand breaks in DNA. As a result
the cell’s DNA repair mechanisms are exploited to include
gene knockouts, integrations or modifications (Hansen et al,
2012). Dr Christina Smith (Sigma-Aldrich, USA) described
a range of products for the analysis and manipulation of
miRNAs.

Dr Tom Quick (Carl Zeiss, UK) presented information about
their PALM MicroBeam Laser micro-dissection for isolating
high-purity tissue. The specimen of interest, typically
a single cell, is isolated without contact, hence contamination
of the sample is prevented and neighbouring tissues/
cells remain unchanged. The genetic and proteomic material
of both the specimen of interest and adjoining areas are
sustained enabling further DNA, RNA and protein analysis
(Micke et al, 2005). This technique can also be used to isolate
live cells which can be successfully re-cultured.

Dr Annaleen Vermeulen (Thermo Fisher Scientific, USA)
explained that technical reproducibility between PCR replicates
in a pooled shRNA screen are significantly improved
by ensuring amplification remains within the exponential
phase and that the correct quantity of genomic DNA
is used to sustain the average template copies per shRNA used during library transduction. This enabled identification
of higher reproducibility of biological replicates in screens
with at least 500-fold shRNA representation (Strezoska
et al, 2012).

Concluding Remarks

Results discussed at RNAi2013 are very promising and
strongly suggest that many common limitations of RNAi based
therapies including successful delivery of ncRNA
and reduction of off-target effects could be overcome in
the foreseeable future. Additionally, RNAi-based therapies
have been developed to specifically target chemoresistant
cancers, DMD, HCV and influenza, amongst many others,
which shows great reassurance for development of personalised
medicines; attendees were encouraged to continue their
work in this field in order to achieve this objective. Data presented
continue to provide hope that RNAi-based therapies
will revolutionise future treatment of disease. Indeed, in the
words of one speaker at the conference, RNAi looks set to
become the next treatment modality.